Method of constructing masses of myocardial cells and use of the myocardial cell mass

ABSTRACT

The object of the present invention is to improve the post-transplantation engraftment rate of cardiomyocytes that have been purified to such an extent that they are free from non-cardiomyocytes and any components derived from other species. 
     To solve this problem, the present inventors studied the possibility of constructing cell masses from the purified cardiomyocytes. As a result, they revealed that the stated problem could be solved by providing a method of preparing cell masses of cardiomyocytes derived from pluripotent stem cells, characterized in that cell masses of aggregated cells containing cardiomyocytes that had been differentiated and induced from pluripotent stem cells were dispersed to single cells to thereby obtain purified cardiomyocytes, which were then cultured in a culture medium under serum-free conditions so that they were reaggregated.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is the National Stage of International Application No.PCT/JP2008/064168, filed Jul. 31, 2008, and claims benefit of JapaneseApplication Nos. JP 2007-200246, filed Jul. 31, 2007, and JP2008-046772, filed Feb. 27, 2008, both of which are herein incorporatedby reference in their entirety.

TECHNICAL FIELD

The present invention relates mainly to a method of preparing cellmasses by aggregating purified cardiomyocytes derived from pluripotentstem cell obtained by dispersing to single cells, as well as a method oftreating cardiac disease by causing the prepared cell masses ofcardiomyocytes to be engrafted in the cardiac tissue, and a method ofpreparing sheets of cell masses using the cell masses of cardiomyocytes.

BACKGROUND ART

Cardiomyocytes in adults have lost the proliferating activity andcardiac transplantation is the only way to treat serious cardiacdiseases such as myocardial infarction and cardiomyopathy. In fact,however, owing to a problem of the lack of cardiac tissue donors, thereis a pressing need to develop a method of treatment other than cardiactransplantation.

In contrast, use of cardiomyocytes prepared outside the living body tosupply with them part of the diseased cardiomyocytes is anticipated tobecome the most promising way to save patients who have to depend oncardiac transplantation. This approach of treatment is called celltherapy on the heart. To bring this therapy into reality, various trialsand errors have been conducted. The methods under review include: usingcardiomyocytes or skeletal myoblasts and bone marrow cells or the likethat have been extracted from fetuses, neonates or adults; usingdifferentiated embryonic stem cells; and obtaining the stem cells (suchas somatic stem cells) which are suggested to exist in the living bodyhas been suggested, and inducing their differentiation (Non-PatentDocument 1: Zhonghua Yi Xue Za Zhi 2003, 83, 1818-22).

These methods can be divided into two approaches. One approach involvestransplanting cardiomyocytes as cells and in this method, thecardiomyocytes dispersed to single cells are directly injected into atissue via injection needle (this method is hereinafter referred to asan “injection method”). The other approach involves constructing atissue or an organ outside the living body (which is hereinafterreferred to as a “tissue engineering method”) and this artificial tissueor organ is transferred into the body for treatment.

Various attempts have been made to implement the tissue engineeringmethod and they include: 1) a method in which cardiomyocytes are formsin sheet-like structure, which are then attached onto a tissue(Non-Patent Document 2: Circulation Research 2002, 90(3):e-40); 2) amethod in which cardiomyocytes and non-cardiomyocytes are mixed in thesame proportions as they are in the cardiac tissue and athree-dimensional structure formed of the mixture is used to replace thetissue; 3) a method in which a three-dimensional structure is formed ofthe cardiomyocytes dispersed to single cells, with a vascular structurebeing further constructed, and the three-dimensional structure issubstituted for the tissue; and 4) a method in which, rather thanreplacing the cardiac tissue, a new auxiliary organ that assists in theinherent organ function is transplanted to a site ectopically(Non-Patent Document 3: Circulation Research 2007 2, 100: 263-272).

However, at the present stage where various trials and errors are underway toward clinical therapeutic application, no method has yet exhibitedpractical data. This is because transplanting cardiomyocytes to theheart involves several problems, such as the inclusion of cells otherthan cardiomyocytes, low engraftment rate of the transplantedcardiomyocytes, and the inability to exclude components derived fromother species.

To use cardiomyocytes as cell masses in transplantation, methods areknown that are capable of constructing cell masses including fetal orneonatal rodent cardiomyocytes and, according to a recent report, cellmasses were constructed using whole cells (including non-cardiomyocytes)that were derived from the fetal heart (Non-Patent Document 4:Developmental Dynamics 235; 2200-2209, 2006). As regards thetransplantation of cardiomyocytes, a case has been reported where fetalmouse cardiomyocytes were transplanted into the hearts of adult mice,which were confirmed to be engrafted (Non-Patent Document Science 1994,264(5155): 98-101). However, this method of the transplantation ofcardiomyocytes involved the use of whole cells to which the whole fetalhearts were dispersed by means of collagenase, so the transplanted cellswere composed of a cell population comprising a mixture ofcardiomyocytes and non-cardiomyocytes. It is also known thatnon-purified cardiomyocytes derived from the living body can betransplanted to the heart (Non-Patent Document 5: Science 1994,264(5155): 98-101; and Non-Patent Document 1: Zhonghua Yi Xue Za Zhi2003, 83, 1818-22).

Also known is a method in which, in the process of differentiation ofembryoid bodies from ES cells, the embryoid bodies are incompletelytreated with a proteolytic enzyme, whereupon a population comprisingcell masses that are rich in cardiomyocytes and those which are not isobtained and then is subjected to density gradient centrifugation,thereby obtaining cell masses that contain up to about 70% ofcardiomyocytes (Patent Document 1: US 2005-0214938 A).

However, each of those methods involves the use of a cell populationthat also contains cells other than cardiomyocytes and contamination ofsuch cells other than cardiomyocytes may have the potential to causeserious unpredictable side effects that may threaten the life of apatient after transplantation. Under the circumstances, it is considerednecessary that cardiomyocytes to be subjected to transplantation therapyshould be used after purification.

Several reports have described achievements in transplanting unpurified,ES cell-derived cardiomyocytes to the heart and allowing them to beengrafted thereafter (Non-Patent Document 6: Cardiovasc Res. 2007 May17; Non-Patent Document 7: Stem Cells. 2007 May 31; and Non-PatentDocument 8: FASEB J. 2007 Apr. 13). According to a recent paper,however, which discussed purifying ES cell-derived cardiomyocytes andinjecting them into the heart, the engraftment rate of the transplantedcardiomyocytes was extremely low and no cardiomyocytes were found to beengrafted (i.e., those survived within the host organ and remainedadherent in it for an extended period of time); as it turned out, thepurified, ES cell-derived cardiomyocytes were not able to be engraftedafter they were transplanted into an individual (the living body)(Non-Patent Document 9: J Exp Med. 2006; 203:2315-27.)

This report has brought light to the difficulty in causing purifiedcardiomyocytes to be engrafted after transplantation. In order to solvethis problem, a method was discovered in the same report that involvedtransplanting the ES cell-derived cardiomyocytes in admixture with mouseembryonic fibroblasts with a for the purpose of enhancing theirengraftment rate after transplantation (Non-Patent Document 9: J ExpMed. 2006 Oct. 2; 203(10): 2315-27). This shows that no known methodsare capable of transplanting purified ES cell-derived cardiomyocytes toremain engrafted while retaining their purity.

In addition, in order to prepare cell transplants that are intended foruse in therapy on the human body, serum and other factors that arederived from other animals must be excluded. In the method of preparingcardiomyocytes to be used in transplantation, culture is usuallyperformed in the presence of serum; but it is known that underserum-free conditions, human ES cells can form embryoid bodies, whichcontain cardiomyocytes in comparable amounts to those obtained by theusual culture in the presence of serum (Non-Patent Document 10: Stemcells and development 15:931-941, 2006). However, no known reportsincluding this report have described a case of transplantingcardiomyocytes that were prepared without using factors such as serumthat were derived from other animals.

Thus, in order that the cardiomyocytes could be successfullytransplanted to the heart, several problems, such as the inclusion ofcells other than cardiomyocytes, the low engraftment rate oftransplanted cardiomyocytes and the inability to exclude componentsderived from other species, must be solved altogether.

Further, in connection with their transplantation to the cardiac tissue,it is contemplated to transplant cardiomyocytes in the form of so-called“cell sheets”. As regards the preparation of cell sheets, it is knownthat neonatal cardiomyocytes are used to form a singlelayered sheet andup to three of such sheets can be stratified in vitro (Non-PatentDocument 11: FASEB J. 2006 April; 20(6): 708-10). However, this documentalso states that, on account of limited oxygen permeability, the cellsheets cannot be made any thicker without neovascularization to the cellsheet, and it is not possible yet to prepare a desired cell sheet thatfits the size of the diseased tissue of the heart.

As described above, the stated of the art is such that the preparationof cardiomyocytes to be used in transplantation and the transplantationof those cardiomyocytes need further improvements from the viewpoint ofpractical feasibility.

Patent Document 1: US 2005-0214938 A

Non-Patent Document 1: Zhonghua Yi Xue Za Zhi 2003, 83, 1818-22

Non-Patent Document 2: Circulation Research 2002, 90(3):e-40

Non-Patent Document 3: Circulation Research 2007 2, 100: 263-272

Non-Patent Document 4: Developmental Dynamics 235; 2200-2209, 2006

Non-Patent Document 5: Science 1994, 264(5155): 98-101

Non-Patent Document 6: Cardiovasc Res. 2007 May 17 (Flk1(+) cardiacstem/progenitor cells derived from embryonic stem cells improve cardiacfunction in a dilated cardiomyopathy mouse model)

Non-Patent Document 7: Stem Cells. 2007 May 31 (Differentiation in vivoof Cardiac Committed Human Embryonic Stem Cells in Post-MyocardialInfarcted Rats)

Non-Patent Document 8: FASEB J. 2007 Apr. 13 (Identification andselection of cardiomyocytes during human embryonic stem celldifferentiation)

Non-Patent Document 9: J Exp Med. 2006 Oct. 2; 203(10): 2315-27

Non-Patent Document 10: Stem Cell and Development 15: 931-941, 2006

Non-Patent Document 11: FASEB J. 2006 April; 20(6): 708-10

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

Hence, the present inventors studied the essential conditions that areassumed at least at present for clinical use of cultured cardiomyocytesand found, as a result, the following problems.

-   (1) Purification of cardiomyocytes: When cardiomyocytes are to be    obtained from the living body or pluripotent stem cells, safety    cannot be secured if they are contaminated by unknown cells, so in    whichever case, it is essential that the cardiomyocytes be highly    purified. In order to maintain the purity of such highly purified    cardiomyocytes in a consistent manner, it would be necessary that    the cardiomyocytes obtained from the living body or pluripotent stem    cells be dispersed to discrete cells (as single cells), the    individual cells being distinguished from one type to another so    that only the cardiomyocytes can be selected.-   (2) Origin of cardiomyocytes: Differences in the properties of    cardiomyocytes that exist between species not only cause the problem    of immune rejection and ethical problems but also present serious    influences on clinical safety and efficacy, so it is important to    use donor cells originating from the same species as the recipient    individual.-   (3) Exclusion of factors derived from animals of other species: To    avoid immunogenicity and contamination by unknown pathogens,    contaminants, such as serum, that are derived from animals of other    species must be excluded.-   (4) Engraftment of transplanted cardiomyocytes: Transplanted    cardiomyocytes are required to function in the same manner as the    cardiomyocytes in the host but, in the first place, they must be    engrafted in the host cardiomyocytes (kept engrafted for an extended    period of time).-   (5) The cells must mature (grow to a bigger size) at the next stage.

In short, the object of the present invention is to provide a means bywhich cardiomyocytes that have been purified to such an extent that theyare free from non-cardiomyocytes and any components derived from otherspecies can be transplanted with an improved engraftment rate thatpromotes maturation.

Means for Solving the Problems

In order to improve the engraftment rate of cardiomyocytes that werederived from the living body or pluripotent stem cells and which werepurified to such an extent that they were free from non-cardiomyocytesand any components derived from other species, the present inventorsstudied the possibility of constructing cell masses from the purifiedcardiomyocytes. As a result, we revealed that the object stated abovecould be solved by providing a method of preparing cell masses ofcardiomyocytes derived from pluripotent embryonic stem cells (ES cells)or induced pluripotent stem cells (iPS cells), characterized in thatcell masses containing cardiomyocytes that had been differentiated andinduced from ES cells or iPS cells were dispersed to single cells tothereby obtain purified cardiomyocytes, which were then cultured in aculture medium under serum-free conditions so that they werereaggregated.

The inventors of the present invention first used cardiomyocytes derivedfrom the living body and studied the possibility of solving theabove-stated object. To be more specific, using the cardiomyocytesderived from the living body that had been purified to such an extentthat they were free from non-cardiomyocytes and any components derivedfrom other species, the present inventors studied the possibility ofenhancing the engraftment rate of such cardiomyocytes aftertransplantation.

As it turned out, the purified cardiomyocytes derived from the livingbody were not capable of forming cell masses even after the lapse of 24hours culturing in a culture medium containing 10% serum. This resultstrongly suggested that the known method of forming cell masses ofcardiomyocytes, as derived from the living body, that was effected in anunpurified state was strongly dependent on the auxiliary action ofnon-cardiomyocytes. In a further experimental study conducted to see howthe purified cardiomyocytes derived from the living body would behaveunder serum-free conditions, those cells were unable to construct cellmasses; on the contrary, they themselves underwent cell death. Theseresults led to the conclusion that “constructing cell masses ofunpurified cardiomyocytes derived from the heart in the living body” isan improbable technique and that it is even impossible to predict thebehavior, in a serum-free condition, of purified cardiomyocytes derivedfrom the heart in the living body.

Next, in the known methods of preparing cell masses of aggregatedcardiomyocytes, cells derived from the heart in neonatal or fetalanimals were used, which were cultured in a serum-containing culturemedium. This is based on the common recognition that sera generally havea strong protective effect, irrespective of the cell species. However,as mentioned before, if therapy is performed on the human body, the useof factors derived from other animals (such as serum) must be avoided.Hence, using serum-free media that were supplemented with variousadditives containing serum substitutes rather than serum, the presentinventors studied the aggregating ability of the cardiomyocytes derivedfrom the living body. However, under any of the serum-free conditionstested, the cardiomyocytes derived from the living body were unable toconstruct the desired cell masses and their survival rate was also poor.

Given a report stating a known method that, when cardiomyocytes derivedfrom the living body were cultured as cell masses, the survival of thecardiomyocytes was maintained for an extended period of time, thepresent inventors made an attempt to form cell masses of purifiedcardiomyocytes, as derived from the living body, under serum-freeconditions. As it turned out, however, no cell masses could be formedeven after five days of culture. This experimental result gave the newfinding that, under serum-free conditions, the purified cardiomyocytesderived from the living body were not capable of constructing cellmasses.

Hence, the present inventors speculated that it might be possible toovercome this technical difficulty by changing the source of supply ofcardiomyocytes. As a result of making similar studies with varioussupply sources, the present inventors revealed that cardiomyocytesderived from purified embryonic stem cells bound together rapidly enoughin only 12 hours even under serum-free conditions, wherebythree-dimensional cell masses of cardiomyocytes could for constructed,and that they had already started to contract synchronously at thattime.

This shows that cardiomyocytes derived from embryonic stem cells werecapable of efficient construction of cell adhesion between purifiedcardiomyocytes and it also shows that the purified cardiomyocytesderived from embryonic stem cells have high ability to aggregate underserum-free conditions. Since this finding was reproduced with more thanone species using embryonic stem cells derived from the respectivespecies, the feature described above would be a nature that is common tothe cardiomyocytes obtained from human and otherwise derived embryonicstem cells. Based on the nature specific for this embryonic stem cellunder serum-free conditions, the present inventors successfully formedcell masses of purified cardiomyocytes under serum-free conditions forthe first time in the art.

Further, it was predicted from a known report that embryonic stemcell-derived cardiomyocytes having low engraftment rate aftertransplantation would also have low capacity to reaggregate(Transplantation 70:1310-1317, 2000); on the other hand, it had beenpredicted that the very attempt to aggregate the purified cardiomyocytesderived from embryonic stem cells would be difficult to realize, makingone believe that they could not be aggregated. Nevertheless, contrary tothis prediction, the present inventors showed for the first time in theart that the purified cardiomyocytes derived from embryonic stem cellscould construct cell masses without the aid of other cells and that asignificant improvement in cell engraftment rate could be achieved byusing the constructed cell masses in transplantation. Briefly, thepresent inventors successfully found out that the cardiomyocytes derivedfrom embryonic stem cells had entirely different characteristics fromthe cardiomyocytes derived from the heart in the living body in thatthey could be purified to single cells and that they could constructcell masses even under serum-free conditions.

Furthermore, with a view to finding out a method by which cell massesmore suitable for transplantation could be prepared from thecardiomyocytes derived from embryonic stem cells, the present inventorsstudied additives that might be added to culture media as a means bywhich the cardiomyocytes derived from embryonic stem cells couldconstruct cell masses more efficiently under serum-free conditions. As aresult, when insulin, transferrin and selenium (ITS) were added as theadditives, the cell masses showed a stronger spontaneous pulsation thanthe cell masses to which no ITS had been added and this phenomenon wasobserved with good reproducibility. Briefly, the addition of ITS (inparticular, insulin) was shown to be desirable for constructing cellmasses using the purified cardiomyocytes derived from embryonic stemcells. Note that insulin is the most important factor among ITS, withtransferrin and selenium playing an auxiliary role.

In the next place, the present inventors added ITS to a serum-free basalculture medium (hereinafter referred to as the basal culture medium) andfurther added a basic fibroblast growth factor (bFGF) and/or aninsulin-like growth factor 1 (IGF1) to the basal culture medium; as itturned out, when bFGF alone was added, the cell masses at day 5 aftertheir formation had a significant increase in diameter, indicating thatthe cells of interest were protected. This phenomenon was not observedunder the serum-containing culture conditions and it was cell protectingand proliferating effects that were characteristic of the serum-freeconditions under which bFGF alone was added. It was known that bFGF hada cell protecting action, a cell growth promoting action and the like oncardiomyocytes being cultured under such experimental conditions thatplane culture was performed in the presence of supplemented serum (J MolCell Cardiol. 2007 January; 42(1): 222-33; and Cardiovasc Res. 2004;64:516-25), but it was entirely unknown that prolonged growth andprotection of cardiomyocytes should occur under serum-free conditions.

This effect was greater than what was observed when cell masses wereformed using a culture medium supplemented with 10% serum; on the otherhand, in the case where bFGF was added but cell masses were not formedby plane culture, the effect of interest was smaller than what wasobserved when cell masses were formed using the culture mediumsupplemented with 10% serum. From the results of these two experiments,it was assumed that, in order to ensure that bFGF would produce the cellprotecting and growth promoting effects in excess of those obtained byforming cell masses using the culture medium supplemented with 10%serum, it was essential to construct the cell masses.

Hence, in order to confirm whether the construction of cell masses wasan essential condition for those effects to be displayed, the presentinventors performed plane adhesive culture of cardiomyocytes derivedfrom purified mouse embryonic stem cells, with bFGF added to the culturemedium, and analyzed the effects of the added bFGF.

In plane culture where no cell masses formed, the purifiedcardiomyocytes could hardly survive in such an environment that ITS wasadded to the basal culture medium. However, upon addition of bFGF, morecells were found to survive and adhered to the culture plate. Thisaction was smaller than the effect that was observed when cell masseswere formed using the culture medium supplemented with 10% serum.

In the case of constructing cell masses of purified cardiomyocytesderived from embryonic stem cells, the action of condition ofserum-free+ITS+bFGF was compared with the action of 10% serum afterlong-time culture; in the 10% serum-containing culture medium, the sizeof cell masses decreased significantly whereas it increasedsignificantly in the serum-free+ITS+bFGF group. This indicated that theaction of bFGF as discovered by the present inventors was thesynergistic effect of all three elements, purification, serum-free, andcell masses.

From this result, the present inventors found that the addition of ITSand/or bFGF to the serum-free culture medium allowed the cell masses ofcardiomyocytes to maintain their state for an extended period of time.By virtue of this finding, the survival rate of cardiomyocytes could beincreased to 90% or more, an outstanding improvement over the 60-70%value for the conventional plane culture method.

Before the accomplishment of the present invention, cell masses ofcardiomyocytes could not be constructed under serum-free and high-purityconditions; however, on the basis of the foregoing, the presentinventors overcame this difficulty by using cardiomyocytes derived fromembryonic stem cells; we also found that cell masses of cardiomyocytescould be constructed most efficiently in the presence of added ITS andbFGF.

Thus, in one of the embodiments, the present invention also provides amethod of preparing cell masses of cardiomyocytes derived from embryonicstem cells, characterized in that purified cardiomyocytes derived fromembryonic stem cell obtained by dispersing aggregated cell masses thatcontain cardiomyocytes differentiated and induced from embryonic stemcells to single cells are cultured in a culture medium under serum-freeconditions so that they are reaggregated. The culture medium to be usedfor the culture described above desirably supplemented with at leastinsulin among ITS, and in a more desirable embodiment, bFGF may also besupplemented. In the method described above, cell masses dispersed tosingle cells need not to be cultured in a single space as in the knownmethods but they may be divided into 10,000 cell groups at maximum andcultured in a corresponding number of independent spaces.

If contaminated by proliferative non-cardiomyocytes, cell masses ofaggregated cells will grow to an extremely large size and also change inmorphology. In addition to this index, staining with a fluorescence dyethat accumulates in mytochondria may be used to identify theproliferative cells as ones in which the dye finds difficultyaccumulating. Cell masses contaminated by such non-cardiomyocytes may berejected from use in transplantation therapy or the like so as to ensurethat the slightest contamination by proliferative non-cardiomyocytes isexcluded from implanted cells.

In addition, it was previously reported that purified cardiomyocytesderived from embryonic stem cell as dispersed to disaggregated cells(single cells) were not engrafted if transplanted as such to the cardiactissue in an individual (the living body) and the present inventorsobtained the same result. In the prior art, this problem was solved bymixing the purified cardiomyocytes derived from embryonic stem cell withauxiliary cells, clearly showing that the protective action ofnon-cardiomyocytes in the living body is essential to the survival ofcardiomyocytes. However, contamination of non-cardiomyocytes canpotentially cause serious unpredictable side effects that might threatenthe life of a patient after transplantation, so the present inventorsspeculated that greater safety and a higher therapeutic effect would besecured if the purified cardiomyocytes could be transplanted into anindividual (the living body) without mixing them with auxiliary cellsbut keeping them at high purity.

Hence, the present inventors got the idea of exploiting the cell massesof purified cardiomyocytes derived from embryonic stem cell obtained bythe method described above and found that by transplanting those cellmasses to the cardiac tissue of an individual (the living body), theengraftment rate after transplantation could be significantly improved.In other words, the present inventors found that, when the purifiedcardiomyocytes derived from embryonic stem cell obtained by dispersingcell masses to single cells were cultured in a culture medium underserum-free conditions so that they were reaggregated to form cellmasses, the engraftment rate of the purified cardiomyocytes derived fromembryonic stem cell after transplantation could be significantlyimproved.

Hence, another embodiment of the present invention relates to a methodof treating cardiac disease, characterized in that cell masses obtainedby reaggregating cardiomyocytes that are derived from embryonic stemcells and which have been purified by dispersing to single cells aretransplanted to the cardiac tissue (especially, a diseased part of thecardiac tissue) in an individual (the living body) such that they areengrafted. The term “engraftment” as used herein means surviving withinthe host organ and remaining adherent in it for an extended period oftime.

Further, as described above, there was known a method that allowed up tothree mono-layered sheets of neonatal cardiomyocytes to be stratifiedbut no thicker sheets of cardiomyocytes could be prepared.

To solve this problem, the present inventors got the idea of exploitingthe cell masses of purified cardiomyocytes derived from embryonic stemcell obtained by the method described above. The cell masses of interestwere constructed by the method described above and the obtained cellmasses were recovered and seeded on the surface of a wall-partitioned,non-cell-adhering vessel with no space between cell masses such thatadjacent cell masses would be continuously in contact with each other,followed by suspension culture. As a result, the cell masses wereconjugated together over time to form a sheet of cell masses ofcardiomyocytes in a thickness of 50-300 μm; it was thus found that aso-called “cell sheet” having a greater thickness than the limit of theprior art could be prepared outside the living body. Thus, it becameclear that in actual modes of application, a desired number of cellmasses in a desired size of purified cardiomyocytes derived fromembryonic stem cells could be used to prepare a cell sheet of a desiredsize.

Hence, a further embodiment of the present invention relates to a methodof preparing a sheet of cell masses of cardiomyocytes (cell sheet),characterized in that cell masses of purified cardiomyocytes derivedfrom embryonic stem cells are subjected to suspension culture as theyare placed at close intervals in the same plane and that the suspensionculture is performed until the cell masses are conjugated together tohave a desired thickness between 50 and 300 μm.

The present invention has revealed that the cell masses of purifiedcardiomyocytes derived from embryonic stem cell having the featuresdescribed above can be transplanted to the cardiac tissue such that theyare engrafted. These cell masses can be used as a medical device fortransplantation that can be transplanted into animal bodies includingthe human body.

Hence, in a further embodiment of the present invention, there isprovided a medical device comprising cell masses of cardiomyocytesderived from embryonic stem cell that have been prepared by a methodwhich comprises preparing cell masses of aggregated cells that containcardiomyocytes differentiated and induced from embryonic stem cells,dispersing the cell masses to single cells to thereby yield purifiedcardiomyocytes derived from embryonic stem cell, and culturing thecardiomyocytes in a culture medium under serum-free conditions so thatthey are reaggregated. This medical device is intended for use intransplantation such that it is transplanted to the cardiac tissue of anindividual such that it is engrafted; it exhibits a significant effectin that it can be applied to a patient who needs cardiactransplantation.

Thus, the present inventors made intensive studies on culture conditionsthat would allow for a significant improvement in the survival rate ofcardiomyocytes derived from embryonic stem cell that had been dispersedto disaggregated cells (single cells) to become completely purified; asa result, we found that the cardiomyocytes had such a new characteristicthat they aggregated to form cell masses when they were cultured in aculture medium under conditions containing no animal-derived serum(i.e., serum-free conditions), preferably in the culture mediumcontaining insulin, more preferably in the culture medium containingtransferrin, selenium, and/or a basic fibroblast growth factor inaddition to insulin. The present inventors transplanted those cells tothe cardiac tissue of an individual (living body) and obtained a newfinding that their engraftment rate in the tissue was significantlyimproved. The present inventors obtained another new finding that, usingthose cells, we could obtain a sheet of cell masses of cardiomyocyteshaving a greater thickness than those expected from the known technique,as well as a medical device comprising those cell masses; these findingsled to the accomplishment of the present invention.

Those findings were obtained by culturing embryonic stem cells andsimilar findings can also be obtained by using other pluripotent stemcells instead of the embryonic stem cells. To state more specifically,when pluripotent stem cells that had been dispersed to a suspension ofdisaggregated cells (single cells) to become completely purified werecultured in a culture medium under conditions containing noanimal-derived serum (i.e., serum-free conditions), preferably in theculture medium containing insulin, more preferably in the culture mediumcontaining transferrin, selenium, and/or a basic fibroblast growthfactor in addition to insulin, those cells were able to aggregate toform cell masses, and the survival rate of cardiomyocytes could beimproved considerably. The pluripotent stem cells that can be usedinclude not only embryonic stem cells but also all other pluripotentstem cells having traits similar to those of embryonic stem cells, asderived from the cells in adult organs and tissues in mammals, as wellas their bone marrow cells, blood cells, and even embryonic and fetalcells; examples are embryonic germ cells (EG cells), germline stem cells(GS cells), and induced pluripotent stem cells (iPS cells).

Thus, the present invention relates to the following matters.

-   (1) A method of preparing cell masses of cardiomyocytes derived from    pluripotent stem cells, characterized in that purified    cardiomyocytes derived from pluripotent stem cell obtained by    dispersing aggregated cell masses that contain cardiomyocytes    differentiated and induced from pluripotent stem cells (such as    embryonic stem cells, embryonic germ cells, germline stem cells or    induced pluripotent stem cells) to single cells are cultured in a    culture medium under serum-free conditions so that they are    reaggregated.-   (2.) The method according to (1) above, wherein the culture medium    contains insulin.-   (3) The method according to (1) or (2) above, wherein the culture    medium contains at least one substance selected from the group    consisting of transferrin, selenium, a basic fibroblast growth    factor (bFGF), an epidermal growth factor (EGF), a platelet-derived    growth factor-BB (PDGF-BB), and endothelin-1 (ET-1).-   (4) The method according to any one of (1) to (3) above, wherein the    content in the culture medium is 0.1 to 10 mg/L of insulin, 0.1. to    10 μg/L of transferrin, 0.1 to 10 μg/L of selenium, 1 ng/ml to 100    ng/ml of the basic fibroblast growth factor, 1 ng/ml to 1000 ng/ml    of the epidermal growth factor, 1 ng/ml to 1000 ng/ml of the    platelet-derived growth factor, and 1×10⁻⁸ to 1×10⁻⁶ M of    endothelin-1 (ET-1).-   (5) A method of treating cardiac disease, characterized in that cell    masses obtained by reaggregating cardiomyocytes that are derived    from purified pluripotent stem cells dispersed to single cells are    transplanted to the cardiac tissue of an individual such that they    are engrafted.-   (6) The method according to (5) above, wherein the cell masses of    cardiomyocytes are those that are obtained by the method according    to any one of (1) to (4) above.-   (7) The method according to (5) or (6) above, wherein the    transplantation comprises injecting the cell masses of    cardiomyocytes into the cardiac tissue.-   (8) The method according to (5) or (6) above, wherein the    transplantation comprises transplanting a sheet of cell masses of    cardiomyocytes onto the cardiac tissue.-   (9) A method of preparing a sheet of cell masses of cardiomyocytes,    characterized in that cell masses of purified cardiomyocytes derived    from pluripotent stem cells are seeded on the surface of a    wall-partitioned, non-cell-adhering vessel, with no space between    cell masses such that adjacent cell masses will be continuously in    contact with each other, followed by suspension culture which is    maintained until the cell masses are conjugated together to have a    desired thickness of 50-300 μm.-   (10) The method according to (9) above, wherein the cell masses of    cardiomyocytes are those obtained by the method according to any one    of (1) to (4) above.-   (11) A medical device comprising cell masses of cardiornyocytes    derived from pluripotent stem cell, for use in transplantation to    the cardiac tissue of an individual such that they are engrafted,    wherein the medical device is prepared by a method comprising    preparing cell masses of aggregated cells that contain    cardiornyocytes differentiated and induced from pluripotent stem    cells, dispersing the cell masses to single cells to thereby yield    purified cardiomyocytes derived from pluripotent stem cell, and    culturing the cardiomyocytes in a culture medium under serum-free    conditions so that they are reaggregated.-   (12) The medical device according to (11) above, wherein the    transplantation comprises injecting the cell masses of    cardiomyocytes into the cardiac tissue.-   (13) The medical device according to (11) above, wherein the    transplantation comprises transplanting a sheet of cell masses of    cardiomyocytes onto the cardiac tissue.

Advantages of the Invention

Discovered by the present invention is a characteristic which describesthat cardiomyocytes derived from pluripotent stem cell that have beenpurified by dispersing to single cells have the ability to reaggregatewhen they are cultured under serum-free conditions. By constructing cellmasses by the method of the present invention, the cardiomyocytes ofinterest can be cultured for an extended period of time with theirsurvival rate or growth capacity being maintained at high level. Inaddition, when those cardiomyocytes were transplanted to the cardiactissue of an individual (the living body), their engraftment rate in thetissue was found to significantly increase and they remained engraftedwithin the cardiac tissue for an extended period without mingling withother cells. This technique has given feasibility to a treatment methodthat holds promise in cell therapy, and a medical device that comprisescell masses of cardiomyocytes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a method of constructing cell masses of cardiomyocytesusing purified cardiomyocytes derived from enhanced green fluorescentprotein (EGFP) expressing mouse embryonic stem cells, and cell massesobtained by using from 313 to 10000 purified cardiomyocytes derived fromthe mouse embryonic stem cells.

FIG. 2 shows the construction of cell masses of cardiomyocytes usingpurified cardiomyocytes derived from marmoset embryonic stem cells,either 24 hours (FIG. 2A) or 48 hours (FIG. 2B) after dispensation.

FIG. 3 shows the results of plane culture of purified cardiomyocytesderived from mouse embryonic stem cells, in particular, theidentification of an optimum adhesive substrate for use in plane culturebased on comparison of the cell survival rate by adhesive substrates(FIG. 3A), and the comparison of cell masses and survival rate ofcardiomyocytes of plane culture depending on the presence of serum inthe culture medium (FIGS. 3B and 3C).

FIG. 4 shows method (1) of detecting embryonic stem cells that arecontaminated in the cell masses formed of purified cardiomyocytesderived from mouse embryonic stem cells; the cell masses marked by thered frames were fbund to contain gigantic cell masses as a result ofcell proliferation.

FIG. 5 shows method (2) of detecting embryonic stem cells that arecontaminated in the cell masses formed of purified cardiomyocytesderived from mouse embryonic stem cells; the contaminated andproliferated non-cardiornyocytes could be detected on the basis of aweak fluorescent signal derived from TMRM (a reagent that specificallystained mitochondria), making it clear that, in contrast with cellmasses of normal cardiomyocytes (FIG. 5A), cell masses of abnormalcardiomyocytes (FIG. 5B) were contaminated by proliferatednon-cardiomyocytes.

FIG. 6A shows that, when cultured on a non-cell-adhesive, round-bottom96-well dish, purified cardiomyocytes derived from the neonatal ratheart were not capable of constructing cell masses even after 24 hoursof culture, and FIG. 6B shows that the purified cardiornyocytes derivedfrom the neonatal rat heart, when cultured under serum-free conditions,did not form cell masses but died after the passage of 5 days.

FIG. 7 shows the results of culture on a non-cell-adhesive, round-bottom96-well dish using serum-free media to which ITS, bFGF and various otheradditives were added (FIG. 7A: 12 hours; FIG. 7B: 6 days); the cellprotecting effect and growth promoting activity of those additives weredetected by revealing effects which they had on the increase in thediameter of cell masses (FIG. 7C).

FIG. 8 shows the results of plane adhesive culture of purifiedcardiomyocytes derived from mouse embryonic stem cells on a cell culturedish coated with fibronectin (FIG. 8A: images after 5 days of culture);the cell viability was significantly low in the serum-free+ITS group andthe ITS+bFGF group (FIG. 8B).

FIGS. 9A, 9B, 9C, and 9D show the results of measuring the viability ofcells in tissue of purified, single-cell cardiomyocytes derived frommouse embryonic stem cells after they were transplanted to the heartwithout applying the reaggregation method but as they remained asdispersed cells.

FIGS. 10A, 10B,and 10C show that, when purified cardiomyocytes derivedfrom mouse embryonic stem cells as formed into cell masses by thereaggregation method were labeled with a red dye (Mitotracker Red) andtransplanted to the heart, the cardiomyocytes survived efficiently.

FIG. 11 shows the results of transplantation to the heart of cell massesof purified cardiomyocytes derived from EGFP expressing mouse embryonicstem cells after reaggregation; FIGS. 11A and 11B show the result ofanalyzing the survival rate of the cells in the cardiac tissue bymeasuring the cell count in the cardiac tissue, and FIGS. 11C and 11Dshow that the cell masses of mouse cardiomyocytes remained engrafted inthe host heart for a prolonged period and could mature with the lapse oftime.

FIG. 12 shows the result of preparing cell masses of purifiedcardiomyocytes derived from marmoset embryonic stem cells under aserum-free condition (FIG. 12A), a condition of serum-free andsupplemented with KSR (FIG. 12B), and a condition of serum free andsupplemented with ITS (FIG. 12C).

FIG. 13 shows the preparation of cardiomyocyte sheets of desired sizeshaving desired thicknesses using cell masses of purified cardiomyocytesderived from marmoset embryonic stem cells.

FIG. 14 shows that cell masses of cardiomyocytes derived from purifiedhuman embryonic stem cells could he prepared under a serum-freecondition.

FIG. 15 shows that cardiomyocytes derived from human stem cells thatwere transplanted to the heart of immunodeficient mice could survive inthe cardiac tissue for 2 weeks.

FIG. 16 shows that cardiomyocytes derived from human stem cells thatwere transplanted to the heart of immunodeficient mice could survive inthe cardiac tissue for 5 weeks, as demonstrated by a red dye used totrace the transplanted cells.

FIG. 17 shows that cardiomyocytes derived from human stem cells thatwere transplanted to the heart of immunodeficient mice could survive inthe cardiac tissue for 5 weeks, as demonstrated by a dye (MicrotrackerRed: red color), Mkx 2.5 (watery blue), and an anti-sarcomeric actininantibody (green) that were used to trace the transplanted cells.

FIG. 18 shows that cardiomyocytes derived from human stem cells thatwere transplanted to the heart of immunodeficient mice could survive inthe cardiac tissue for 5 weeks, as demonstrated by Mkx 2.5 (watery blue)and an anti-human antibody (green).

FIG. 19 shows the results of preparing cell masses of purifiedcardiomyocytes derived from mouse iPS cells by culture under aserum-free condition, a condition of scrum-free and supplemented withITS, and a condition of serum-free and supplemented with KSR. FIG. 19Ashows the result of an FACS analysis conducted with the mitochondrialindicator TMRM FIG. 19B show the result of immunostaining (with actininand Nkx2.5) of purified cardiomyocytes that have been subjected toadhesive culture. FIG. 19C shows the appearance of the purifiedcardiomyocytes 24 hours after they were seeded in a non-cell-adhesive96-well culture dish.

FIG. 20 shows the results of culturing purified, human ES cell-derivedcardiomyoeytes under serum-free conditions in the presence of added bFGFor other growth factors (FIG. 20A bFGF was preferential in the cellprotecting and growth activating effects (FIG. 20B).

FIG. 21 shows the expression of genes for hFGF, EGF, PDGE-BB, and ET-1in the host heart as it relates to the mechanism of maturation for thecase where cell masses of mouse cardioniyocytes remain engrafted in thehost heart for a prolonged period of time.

BEST MODE FOR CARRYING OUT THE INVENTION

Those ordinarily skilled in the art who, in order to carry out thepresent invention, needs to know about methods in molecular biology,genetic engineering methods such as recombinant DNA technology, generalmethods in cell biology as well as the prior art may, unless otherwiseinstructed, refer to standard books in those fields. Examples of suchbooks include: “Molecular Cloning: A Laboratory Manual, 3^(rd) Edition”(Sambrook & Russell, Cold Spring Harbor Laboratory Press, 2001);“Current Protocols in Molecular biology” (Ed. by Ausubel et al., JohnWiley & Sons, 1987); “Methods in Enzymology in series” (Academic Press);“PCR Protocols: Methods in Molecular Biology” (Ed. by Bartlett &Striling, Humana Press, 2003); “Animal Cell Culture: A PracticalApproach, 3^(rd) Edition” (Ed. by Masters, Oxford University Press,2000); and “Antibodies: A Laboratory Manual” (Ed. by Harlow et al. &Lane, Cold Spring Harbor Laboratory Press, 1987). The reagents and kitsfor use in cell culture and experiments in cell biology that arereferred to herein are available from commercial suppliers such asSigma, Aldrich, Invitrogen/GIBCO, Clontech, and Stratagene.

(1) Pluripotent Stem Cells

Those ordinarily skilled in the art who, in order to carry out thepresent invention, needs to know about cell culture using pluripotentstem cells and general methods for experiments in developmental and cellbiology may, unless otherwise instructed, refer to standard books inthose fields. Examples of such books include: “Guide to Techniques inMouse Development” (Ed. by Wasserman et al., Academic Press, 1993);“Embryonic Stem Cell Differentiation in vitro” (M. V. Wiles, Meth.Enzymol. 225:900, 1993); “Manipulating the Mouse Embryo: A laboratorymanual” (Ed. by Hogan et al., Cold Spring Harbor Laboratory Press,1994); “Embryonic Stem Cells” (Ed. by Turksen, Humana Press, 2002). Thereagents and kits for use in cell culture and experiments indevelopmental and cell biology that are referred to herein are availablefrom commercial suppliers such as Invitrogen/GIBCO and Sigma.

For the methods of preparing, serially culturing and preserving mouse orhuman pluripotent stem cells, standard protocols have already beenestablished and those ordinarily skilled in the art who wants to carryout the present invention are able to use those pluripotent stem cellsby referring to a plurality of reference documents and the like inaddition to the reference books listed in the preceding sections. Suchdocuments include the following: Matsui et al., Cell 70:841, 1992;Thomson et al., U.S. Pat. No. 5,843,780; Thomson et al., Science 282:114, 1998; Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998;Shamblott et al., U.S. Pat. No. 6,090,622; Reubinoff et al., Nat.Biotech. 18:399, 2000; and International Publication WO 00/27995 A1. Forother animal species, such as monkey (Thomson et al., U.S. Pat. No.5,843,780; and Proc. Natl. Acad. Sci. USA, 92, 7844, 1996), rat(Iannaccone et al., Dev. Biol. 163:288, 1994; and Loring et al.,International Publication WO 99/27076 A1), chicken (Pain et al.,Development 122:2339, 1996; U.S. Pat. Nos. 5,340,740; and 5,656,479),and swine (Wheeler et al., Reprod. Fertil. Dev. 6:563, 1994; and Shim etal., Biol. Reprod. 57:1089, 1997), methods are known that can establishpluripotent cells such as embryonic stem cells and embryonic stemcell-like cells, and those pluripotent stem cells that can be used inthe present invention may be prepared or used in accordance with themethods described in those documents.

The method of the present invention can be applied to pluripotent stemcells derived from any mammals. For example, it may be applied topluripotent stem cells derived from the mouse, bovine, goat, dog, cat,marmoset, rhesus monkey, and human; however, it is not limited to thepluripotent stem cells derived from these animal species. Thepluripotent stem cells to be used in the present invention may beexemplified by embryonic stem cells (ES cells) derived from mammals suchas mouse, monkey and human that are already widely used as culturedcells.

Specific examples of mouse-derived embryonic stem cells include EB3cell, E14 cell, D3 cell, CCE cell, R1 cell, 129SV cell, and J1 cell. Themouse-derived embryonic stem cells according to the present inventionare available from the American Type Culture Collection (ATCC),Chemicon, Cell & Molecular Technologies, etc.

As for the Monkey-derived embryonic stem cells, those cell linesestablished from rhesus monkey (Macaca mulatta) (Thomson et al., Proc.Natl. Acad. Sci. USA 1995; 92:7844), cynomolgus monkey (Macacafascicularis) (Suemori et al., Dev. Dyn. 2001; 222: 273-279) and commonmarmoset (Callithrix jacchus) (Sasaki et al., Stem Cells. 2005; 23:1304-1313) have been reported and are available. For example, marmosetembryonic stem cells are also available from the Central Institute forExperimental Animals (a judicial foundation).

As of today, more than several tens of human derived embryonic stem celllines have been established in the world; for example, in the list atthe US National Institutes of Health(http://stemcells.nih.gov/registry/index.asp), numerous cell lines areregistered for public use, and other cell lines are available from thecommercial sources including Cellartis, ES Cell International, WisconsinAlumni Research Foundation, etc. In Japan, human derived embryonic stemcell lines are also available from the Stem Cell Research Center,adjunct facilities to the Institute for Frontier Medical Sciences, KyotoUniversity (national university corporation) (Suemori et al., Biochem.Biophys. Res. Commun., 2006; 345: 926-932).

It was also reported that embryonic stem cell lines have beenestablished for bovine (Mitalipova et al., Cloning 2001; 3: 59-67),avian (Petitte et al., Mech. Dev. 2004; 121: 1159-1168), and zebrafish(Fishman, M. C., Science 2001; 294: 1290-1291).

While embryonic stem cell lines are generally established by culturingearly embryos, they can also be prepared from early embryos into whichthe nuclei of somatic cells have been transferred (Munsie et al., Curr.Biol. 10:989, 2000; Wakayama et al., Science 292:740, 2001; and Hwang etal., Science 303: 1669, 2004). There have also been reported an attemptto develop parthenogenetic embryos to a stage comparable to theblastocyte stage and to prepare embryonic stem cells from that stage(U.S. patent Publication Ser. No. 02/168,763 A1; and Vrana K et al.,Proc. Natl. Acad. Sci. USA 100:11911-6) and a method in which anembryonic stem cell is fused to a somatic cell to make an embryonic stemcell carrying the genetic information from the somatic cell nucleus(International Publication WO 00/49137 A1; and Tada et al., Curr. Biol.11:1553, 2001). The embryonic stem cells that can be used in the presentinvention also include those that have been prepared by the methodsdescribed above, as well as those in which the genes located on theirchromosomes have been modified by genetic engineering techniques.

The pluripotent stem cells that can be used in the method according tothe present invention are not limited to embryonic stem cells butinclude all other pluripotent stem cells having traits similar to thoseof embryonic stem cells, as derived from the cells in adult organs andtissues in mammals, as well as their bone marrow cells, blood cells, andeven embryonic and fetal cells. In this case, the “traits similar tothose of embryonic stem cells” may be defined by cellular biologicalproperties that are specific to embryonic stem cells, as exemplified bythe presence of a surface (antigen) marker specific to embryonic stemcells, expression of a gene specific to embryonic stem cells, as well asa teratoma forming capacity and chimeric mouse forming capacity.Specific examples of other applicable pluripotent stem cells includeembryonic germ cells (EG cells) prepared from primordial germ cells,germline stem cells (GS cells) prepared from germ cells in the testis,and induced pluripotent stem cells (iPS cells) prepared from somaticcells such as fibroblasts by a special gene manipulation. Examples ofthe induced pluripotent stem cells include those that can be prepared byintroducing specific factors into somatic cells and they can be preparedby the methods descried in a paper written by the research group ofProfessor Shinya Yamanaka at Kyoto University (K. Takahashi, et al.,“Induction of Pluripotent Stem Cells from Adult Human Fibroblasts byDefined Factors” Cell 2007 131: 861-872) and a paper written byThomson's research group at Wisconsin University (J. Yu, et al.,“Induced Pluripotent Stem Cell Lines Derived from Human Somatic Cells”Science 2007 318:1917-1920). Specifically, at least one gene selectedfrom genes of Oct3/4, Sox2, c-Myc, Klf4, Nanog and LIN28 is transferredinto a given somatic cell, the expression of any gene or protein that isspecific for pluripotent stem cells is detected, and those cells thatexpress such gene or protein are selected as pluripotent stem cells.Like embryonic stem cells, the induced pluripotent stem cells thusprepared can be cultured together with a basic fibroblast growth factorin the presence of mouse fibroblasts deactivated for growth or cellsthat can be substituted for them, and the cultured cells can be used aspluripotent stem cells, similar to embryonic stem cells.

It has heretofore been revealed that the induced pluripotent stem cellsdescribed above have the same properties as the embryonic stem cellswith regard to the characteristics of differentiation into varioustissues and those of gene expression within cells (Park I. H. et al.,Nature, 2008, 451, 141-147) and the conditions for inducingdifferentiation of embryonic stem cells into a variety of tissues candirectly be applied to the induced pluripotent stem cells (Takahashi andYamanaka, Saibou Kogaku (Cell Engineering), Vol. 27, No. 3, 252-253,2008).

(2) Methods of Inducing Differentiation of Pluripotent Stem Cells intoCardiomyocytes

The following description relates to embryonic stem cells (ES cells) asan example of pluripotent stem cells. When embryonic stem cells capableof differentiating into cardiomyocytes are subjected to an appropriatetreatment for inducing differentiation into cardiomyocytes, they startto differentiate into cardiomyocytes. For example, differentiation ofmouse embryonic stem cells into cardiomyocytes can be induced by thehanging drop method, in which the embryonic stem cells are subjected tosuspension-culture in a culture media free of a leukemia-inhibitingfactor (LIF) until cell masses (embryoid bodies) are formed.Alternatively, marmoset embryonic stem cells or human embryonic stemcells may likewise be subjected to a treatment for inducingdifferentiation into cardiomyocytes. To induce differentiation ofembryonic stem cells into cardiomyocytes, any known methods may beemployed. For example, a method of inducing differentiation in thepresence of a substance that suppresses BMP signaling (WO2005/033298)and a method of inducing differentiation in the presence of a substancethat stimulates activation of the canonical Wnt signaling pathway(PCT/JP2007/59242, published as WO2007/126077).

(3) Purification of Cardiomyocytes

After inducing the differentiation of embryonic stem cells intocardiomyocytes by the method described in (2) above, the cardiomyocytesmay be purified (selected) by any method that is capable of dispersingcardiomyocytes into disaggregated cell (single cells) and purifying themas individual cardiomyocytes. For example, a method of selection usingmitochondria in cardiomyocytes as an index (WO2006/022377) and a methodof selecting cells that can survive under low nutrient conditions(PCT/JP2007/051563, published as WO2007/088874) may be used to purify(select) only cardiomyocytes.

The purified cardiomyocytes derived from embryonic stem cell that havebeen obtained through dispersing to single cells according to the methoddescribed in (3) above may be cultured under serum-free conditions suchthat they are aggregated to prepare cell masses of cardiomyocytesderived from embryonic stem cells, Preferably, the culture medium usedfor this culture contains at least one substance selected from the groupconsisting of insulin (0.1 to 10 mg/L), transferrin (0.1 to 10 μg),selenium (0.1 to 10 μg/L), a basic fibroblast growth factor (bFGF; 1ng/ml to 100 ng/ml), an epidermal growth factor (1 ng/ml to 1000 ng/ml),a platelet-derived growth factor (1 ng/ml to 1000 ng/ml), andendothelin-1 (ET-1) (1×10⁻⁸ to 1×10⁻⁶ M).

The cell masses of purified cardiomyocytes derived from embryonic stemcell that have been obtained by the method described above containproliferative cells as a small number of contaminant; if suchproliferative cells are excluded from cells for transplantation, furthersafety can be secured. Currently known methods for purifyingcardiomyocytes involve preliminary introduction of certain marker genesinto the genome of the stem cells (FASEB J. 2000; 14: 2540-2548). All ofthese methods can provide 99% purity but they are incapable ofguaranteeing 100±0% purity. For example, if 10¹¹ cardiomyocytes arerequired for treating human myocardial infarction, 99% purity meanscontamination by 10⁹ non-cardiomyocytes. Thus, even a method that may bedescribed as an almost perfect means of purification in light of theknown state of the art does not enable 100% purification ofcardiomyocytes and must be combined with further methods of purificationor applied by other methods that guarantee safety.

Hence, the present inventors replicated the above-described method afterintentionally mixing the cell masses of undifferentiated cardiomyocyteswith embryonic stem cells. As it turned out, the undifferentiatedembryonic stem cells which were more capable of growth thancardiomyocytes constructed separate larger cell masses outside the cellmasses of cardiomyocytes. The cell masses of cardiomyocytes contaminatedby the undifferentiated embryonic stem cells can be clearly detected bychecking the overall sizes of the cell masses. The present inventorsalso added a mitochondrial indicator (e.g., TMRM) to the cell masses ofinterest, whereupon the cardiomyocytes that were rich in mitochondriawere found bright whereas the embryonic stem cells and otherproliferative cells that were not rich in mitochondria were found dark.Exclusion of the cell masses having the greater difference influorescence can be excluded automatically by making use of Arrayscan(Cellomics), Incell 1000 (GE/Amersham Biosciences, Cardiff, UK),Scanalyzer (Scanalyzer LemnaTec, Aachen Germany), “ImageXpress MICRO”(Molecular Devices, Union City, USA), “Pathway HT” (Becton DickinsonBiosciences), “Scan^R” (Olympus Soft Imaging Solutions, Germany), etc.Thus, the method described above provides a simple and automatic way toidentify the contamination by the undifferentiated embryonic stem cells.Briefly, the proliferative cells that slightly mix with the purifiedcardiomyocytes derived from embryonic stem cell that have formed asaggregates into cell masses under serum-free culture conditions can beidentified using the size and shape of such cell masses as indices,which is optionally combined with staining with a mitochondrialindicator and subsequent identification using fluorescence intensity andits distribution within cell masses as indices. In this way, the cellmasses contaminated by non-cardiomyocytes can be excluded from the cellsfor transplantation to thereby achieve greater safety.

(5) Transplantation of Cell Masses of Cardiomyocytes to the CardiacTissue and their Engraftment

Using the cell masses obtained through aggregation by the methoddescribed above, namely, the cell masses of purified cardiomyocytesderived from embryonic stem cells, one can transplant only thecardiomyocytes to the cardiac tissue of an individual (the living body).For example, the cardiomyocytes may be directly injected into thecardiac tissue through a syringe; in this case, injection is feasibleusing a thin (29- or 30-gage), hence, less invasive needle. Theengraftment rate of the cardiomyocytes transplanted by the methoddescribed above is significantly improved over the known methods. Theterm “engraftment” means that the transplanted cells survive within thehost organ and remain adherent inside the organ for an extended periodof time.

(6) Sheets for Transplantation Made of Cell Masses of Cardiomyocytes

By means of known methods, a sheet of cardiomyocytes thicker than threecells thick cannot be prepared at a time even if neonatal cardiomyocytesare used. However, in the present invention, after constructing cellmasses of purified cardiomyocytes derived from embryonic stem cells, theobtained cell masses are recovered, seeded on the surface of awall-partitioned, non-cell-adhering vessel with no space between cellmasses such that adjacent cell masses will be continuously in contactwith each other, and subjected to suspension culture, whereupon, thecell masses of cardiomyocytes are conjugated together over time to forma sheet of cell masses of cardiomyocytes (cell sheet) having a thicknessof 50-300 μm. Hence, culture is performed until a desired thickness isformed. As a result, in actual application modes, cell masses in adesired size of purified cardiomyocytes derived from embryonic stemcells can be used in a desired number to prepare a cell sheet of adesired size.

EXAMPLES

The present invention is illustrated in greater detail by reference tothe following examples.

Example 1 Preparation of Cardiomyocytes Derived from Mouse EmbryonicStem Cells and Purification of the Cardiomyocytes Using the MitochondriaMethod

The purposes of this Example were to prepare cardiomyocytes from mouseembryonic stem cells and to study whether it was possible to purify theprepared cardiomyocytes using a mitochondrial indicator.

As embryonic stem cells, EB3 cell line (Niwa H, et al., Nat Genet. 2000;24: 372-376) was used. An EGFP expressing unit was introduced into theEB3 cell line via a plasmid and EGFP expressing cells were acquired andestablished as a cell line. The thus acquired EGFP-expressing embryonicstem cells (EB3 cells) were suspended in an α-MEM culture medium (Sigma)such that the concentration of embryonic stem cells reached 75 cells/35μL; the α-MEM culture medium was supplemented with heat-inactivatedfetal bovine serum (55° C.×30 min) to a final concentration of 10%.Subsequently, the suspension of mouse embryonic stem cells thus preparedwas distributed in a commercial cell culture 384-well plate (product ofGreiner, Model 788161; i.d. of each well opening, 3.0 mm) and embryoidbodies were prepared in accordance with the following method.

The 384-well plate had a nominal allowable liquid volume of 25 μL perwell but in order to raise the liquid level above the well openings bythe effect of surface tension, the suspension was distributed in avolume of 35 μL per well. As a result, 75 embryonic stem cells weredistributed per well. In this case, the suspension had to be supplied ina volume of 28 μL in order to reach the horizontal level in each openingand in an additional volume of 7 μL to rise above that horizontal level.For distribution of the suspension, a multi-channel pipette of TheremoLabsystems (Lot No. 4610070) or a distributing machine of BioTech Co.,Ltd. (Model LD-01) was used.

The plate in which the culture medium containing the embryonic stemcells was distributed until it raised above the well openings wasinverted upside down so that the culture medium was projecting downwardfrom the lower edges of the well openings. As it was kept in this state,the plate was covered with a lid and culture was performed in anincubator at 37° C. in a 5% CO₂ atmosphere until embryonic stem cellsgrew in the projections from the lower edges of the well openings. Oneday after the start of culture, the plate with the projecting liquidlevel of the culture medium facing down was held with clean tweezers orthe like and the projections of the culture medium were brought intocontact with the surface of an α-MEM culture medium (Sigma) filling aseparate larger vessel that was supplemented with heat-inactivated fetalbovine serum (55° C.×30 min) to a final concentration of 10%; the cellmasses were allowed to precipitate under their own weight into theculture medium in the larger vessel, thereby recovering embryoid bodiesor the cell masses derived from the embryonic stem cells.

The recovered embryoid bodies were cultured in a non-cell-adhesive dish(Asahi Techno Glass, sterile Petri dish #SH90-15; or Eiken Chemical Co.,Ltd., sterile rectangular Petri dish type 2) for an additional 2 or 3days. The cultured embryoid bodies were recovered into a centrifugaltube and after replacing the suspension with a serum-free culture medium(α-MEM culture medium (#MO644 of SIGMA) supplemented with an ITSsolution (GIBCO #41400-045) after 1/100 dilution (the ITS solution usedin the present invention contained 1 g/L of insulin, 0.55 g/L oftransferrin, and 0.67 mg/L of selenium chloride)), the embryoid bodieswere cultured in a cell adhesive, sterile culture dish (FALCON #353003).

Culture medium was changed every other day until the 15^(th) day ofculture for differentiation. To the sample at day 15, a mitochondrialindicator TMRM (Invitrogen #T668) was added at a final concentration of10 mM, which was incubated for 2 hours. Thereafter, using aphysiological buffer (116 mM NaCl, 20 mM Hepes, 12.5 mM NaH₂PO₄, 5.6 mMglucose, 5.4 mM KCl, 0.8 mM MgSO₄, pH 7.35) containing collagenase(Wortington Type 3) and trypsin (DIFCO #215240) each added at a finalconcentration of 0.1%, the cultured cells were dispersed to single cellswith the culture medium being stirred. The sample, or the suspension ofsingle cells, was loaded in a fluorescent activated cell sorter (FACS)to thereby recover highly fluorescent cell groups (WO 2006/022377). Thepurified cells were counted for the numbers of viable and dead cells bymeans of a hematocytometer. As it turned out, the proportion of theviable cells was about 75%.

Example 2 Preparation of Cell Masses Using Cardiomyocytes Derived fromMouse Embryonic Stem Cells

The purpose of this Example was to know whether it was possible toprepare cell masses using the cardiomyocytes derived from mouseembryonic stem cell that were prepared in Example 1.

The purified, cardiomyocytes derived from mouse embryonic stem cell thatwere prepared in Example 1 were distributed in non-cell-adhesive, roundbottom 96-well plates (SUMITOMO BAKELIKE CO., LTD.; CELLFECTIGHTSPHEROID) such that 10,000, 5,000, 2,500, 1,250, 625 or 313 cells wouldbe present in each well. The culture medium was α-MEM supplemented with10% fetal bovine serum. The distributed cells were observed over time;10 hours later, cell masses formed and started to beat spontaneously ina synchronous manner. Twenty-four hours later, the cell masses eachassumed a nearly perfect spherical shape and 10 days later, rhythmic,synchronous and spontaneous beating occurred (FIG. 1).

These results showed that, after the cardiomyocytes derived from mouseembryonic stem cells were dispersed to single cells, they could bereaggregated to form cell masses.

Example 3 Preparation of Cell Masses Using Cardiomyocytes Derived fromMarmoset Embryonic Stem Cells

The purpose of this Example was to know whether it was possible toprepare cell masses using cardiomyocytes derived from marmoset embryonicstem cell that were prepared in accordance with the method of Example 1.

The marmoset embryonic stem cells were obtained from the CentralInstitute for Experimental Animals (Sasaki E, et al., Stem Cells. 2005;23(9): 1304-13). Using mouse embryonic fibroblasts (MEF) that had beengrowth-inactivated by mitomycin C treatment, these marmoset embryonicstem cells were cultured such that they would remain undifferentiated.The culture medium was composed of KO-DMEM (GIBCO), 20% KO-SERUM(GIBCO), 1.6 mM L-glutamine, 0.1 mM non-essential amino acids (MEM), 0.2mM β-mercaptoethanol (2-ME; Sigma), 100 IU/ml penicillin, 100 μg/mlstreptomycin sulfate, and 8 ng/ml each of a recombinant human leukemiainhibiting factor (LIF; Chemicon) and a recombinant human basicfibroblast growth factor (bFGF; Peprotech). For serial passage, coloniesof embryonic stem cells were separated by treatment with 0.1% type IIIcollagenase (Wortington) at 37° C. for 10 minutes.

Subsequently, in order to separate the embryonic stem cells from MEF,the culture medium containing cell masses was passed through a mesh witha pore size of 100 μm, which was then passed through a mesh with a poresize of 40 μm to discard the undersize fraction; the cell masses in theoversize fraction were recovered. The recovered cell masses were thoseof pure embryonic stem cells. For differentiation, 50-1,000 embryonicstem cells per EB were cultured as embryoid bodies on anon-cell-adhesive bacterium dish (Asahi Techno Glass; sterile Petridish) for a total of 15-30 days so that they differentiated intoembryoid bodies including cardiomyocytes. The culture medium used forthis differentiation was the same as identified above, except that itdid not contain bFGF, i.e., it was composed of KO-DMEM (GIBCO), 20%KO-SERUM (GIBCO), 1.6 mM L-glutamine, 0.1 mM non-essential amino acids(MEM), 0.2 mM β-mercaptoethanol (2-ME; Sigma), 100 IU/ml penicillin, 100μg/ml streptomycin sulfate, and 8 ng/ml of a recombinant human leukemiainhibiting factor (LIF; Chemicon).

One or two months after their preparation, the embryoid bodies werepicked up and treated by the method described in WO 2006/022377 topurify the cardiomyocytes. To be more specific, the embryoid bodies weretreated with collagenase and trypsin to give disaggregated single cells.To the culture medium as a cell suspension, a mitochondrial indicatorTMRM (Invitrogen #T66) was added at a final concentration of 10 mM andthe mixture was left to stand at 37° C. for 15 minutes, washed threetimes, and immediately subjected to FACS analysis. Cells(cardiomyocytes) displaying a higher fluorescent intensity than theprincipal cell population were separated and recovered.

The separated cardiomyocytes were treated by the same method as inExample 2 to prepare cell masses of cardiomyocytes. To be more specific,the purified cardiomyocytes derived from marmoset embryonic stem cellswere distributed in a non-cell-adhesive, round bottom 96-well plate(SUMITOMO BAKELIKE CO., LTD.; CELLFECTIGHT SPHEROID) such that 2,000cells would be present in each well. The distributed cells were observedover time; 24 hours later, cell masses formed (FIG. 2A) and started tobeat spontaneously in a synchronous manner. Forty-eight hours later, thecell masses each assumed a nearly perfect spherical shape (FIG. 2B) and10 days later, rhythmic, synchronous and spontaneous beating occurred(FIG. 2).

These results showed that, after the cardiomyocytes derived frommarmoset embryonic stem cells were dispersed to single cells, they couldbe reaggregated to form cell masses.

Example 4 Measurement of Cell Survival Rate for Cell Masses Formed byUsing Cardiomyocytes Derived from Mouse Embryonic Stem Cells andComparison with the Result of Adhesive Culture

The purposes of this Example were to study the adhesive substrate withthe strength of protective action under plane culture conditions beingused as an index, and to compare the survival rate of purified,embryonic stem cell-derived cardiomyocytes between plane adhesiveculture and cell mass culture; the plane adhesive culture was performedusing serum having a strong cell protecting action, and the cell massculture was performed in the condition with or without serum; the cellprotecting action was found to be superior when cell mass culture wasperformed under serum-free conditions.

In Example 4, cardiomyocytes derived from mouse embryonic stem cellswere purified in accordance with Example 1.

The purified cardiomyocytes were seeded in plastic culture dishes(product of BD), coated with either (1) gelatin or (2) fibronectin, inthe presence of serum (FIG. 3A). In addition, with a view to formingcell masses, (3) the purified cardiomyocytes were distributed in anon-cell-adhesive, round bottom 96-well plate in accordance with Example2 such that 1,000 cells would be present in one well and the suspensionswere centrifuged at 120 g for 5 minutes. Further in addition, (4) thepurified cardiomyocytes were distributed in a non-cell-adhesive, roundbottom 96-well plate in accordance with Example 2 except for usingserum-free conditions to construct cell masses, such that 1,000 cellswould be present in each well and the suspensions were centrifuged at120 g for 5 minutes. The samples of (1) to (4) were cultured in the sameincubator for 12 hours and 4 days. Four days later, the number of cellsadhering to each dish (the viable cell count) and that of non-adherentbut suspending cells (dead cell count) were measured. The results aredepicted in FIG. 3 (see FIG. 3A for the conditions of (1) and (2), andalso see FIGS. 3B and 3C for the conditions of (3) and (4),respectively.)

As it turned out, the cell viability in cell mass culture underserum-free conditions was obviously higher than the maximum value forplane adhesive culture in the presence of serum (ca. 60% in the case of(2)), i.e., 99.2% viable in the case of (3) and 90.4% viable in the caseof (4).

Example 5 Preparation of Cell Masses Using Cardiomyocytes Derived fromPurified Mouse Embryonic Stem Cells and Detection of ContaminatedEmbryonic Stem Cells

The purpose of this Example was to detect non-cardiomyocytes that werecontaminated in cell masses formed of purified cardiomyocytes derivedfrom mouse embryonic stem cells.

Cell masses of purified cardiomyocytes were prepared by the methods ofExamples 1 and 2, provided that prior to the final seeding of the96-well plate, 2% of undifferentiated embryonic stem cells were added tothe suspension of cardiomyocytes. The cell masses were cultured in aserum-free α-MEM solution that contained 1 mg/ml of insulin and 10 nM ofTMRM; 14 days later, fluorescent images and phase-contrast images wereacquired from all wells.

As a result, in two wells that accounted for about 2% of the wells, alarger cell mass (more than twice the size of normal cell masses) wasobserved (FIG. 4) and it was found that part of these non-spherical cellmasses formed a cell population that emitted a weak, TMRM-derivedfluorescent signal (i.e., non-cardiomyocytes) and which was composed ofabnormal cardiomyocytes (FIG. 5B). The whole cell masses of normalcardiomyocytes emitted a TERM derived fluorescent signal (FIG. 5A).

Thus, the method provided by Example 5 enabled contaminantnon-cardiomyocytes to be identified with high sensitivity.

Example 6 Preparation of Cell Masses Using Purified CardiomyocytesDerived from Neonatal Rat Heart

The purpose of this Example was to know whether it was possible toprepare cell masses using purified cardiomyocytes derived from neonatalrat heart.

Neonatal rats 0-2 days after birth were anesthetized with ether. Theheart was excised and the cardiac tissue was dispersed intodisaggregated cells with 0.1% collagenase (Wortington). The cells werestained with 10 nM TMRM and then treated by FACS to purify thecardiomyocytes.

The number of the purified cardiomyocytes was counted and cultured in anon-cell-adhesive 96-well dish (SUMITOMO BAKELITE) with 3,000 cellsbeing seeded per well. The culture medium consisted of DMEM-high glucose(Invitrogen) supplemented with 10% FBS (JRH).

The appearance of the cells after 24 hours of culture is depicted inFIG. 6. The purified cardiomyocytes derived from the neonatal rat heartdid not form cell masses but aligned along the round bottom of each well(FIG. 6A). The purified cardiomyocytes derived from the neonatal ratheart at day 5 of culture were virtually dead (FIG. 6B) under theserum-free conditions (solely with the basal culture medium α-MEM (leftpanel of FIG. 6B, or with α-MEM+ITS (right panel of FIG. 6B)).

Example 7 Culture Medium Composition Optimum for Forming Cell MassesUsing Cardiomyocytes Derived from Mouse Embryonic Stem Cells

The purpose of this Example was to analyze the various properties ofneonatal rat's primary cardiomyocytes and cardiomyocytes derived frommouse embryonic stem cells so as to find out a culture medium mostsuitable for the cardiomyocytes derived from embryonic stem cells.

Purified cardiomyocytes derived from mouse embryonic stem cells andpurified neonatal rat cardiomyocytes were prepared as described above;they were then cultured in each of 10 different condition: one wassolely composed of α-MEM and the other nine consisted of α-MEM+ITS,α-MEM+ITS+50 ng/ml bFGF (peprotech), α-MEM+ITS+50 ng/ml IGF-1 (Wako),α-MEM+ITS+50 ng/ml bFGF+50 ng/ml IGF-1, α-MEM+5% KSR (knockout serumreplacement: Invitrogen), α-MEM+10% KSR (knockout serum replacement:Invitrogen), α-MEM+1% FBS (Equitech Bio), α-MEM+5% FBS, and α-MEM+10%FBS, respectively.

When the purified cardiomyocytes derived from mouse embryonic stem cellswere cultured in a non-cell-adhesive, round bottom 96-well dish, cellmasses formed in all culture media in just 12 hours (FIG. 7A). However,the neonatal rat cardiomyocytes failed to form cell masses even after 24hours under all culture conditions. A culture medium supplemented with10% serum, is given as an example. After 4 days, the neonatal ratcardiomyocytes formed cell masses only in the media supplemented with 5%FBS and 10% FBS, respectively.

The cell masses of purified, cardiomyocytes derived from mouse embryonicstem cell that formed after 6 days of culture in a non-cell-adhesive,round bottom 96-well dish were observed (FIG. 7B); a significantincrease in the diameter of cell masses, as compared to that of the cellmasses just formed, was found only in the culture medium of α-MEW+ITS+50ng/ml bFGF (see FIG. 7C, in particular, please refer to the columnmarked with the asterisk). Even in the serum-containing media, thediameter of cell masses was about one half the value for the early stage(FIG. 7C).

From the foregoing, it is believed that the serum-free culture mediumsupplemented with ITS and bFGF has a very strong cell protecting actionand exhibits a unique property of inducing the proliferation ofcardiomyocytes.

Example 8 Culture Medium Composition Optimum for the Formation of CellMasses Using Cardiomyocytes Derived from Mouse Embryonic Stem Cells

In Example 7, it was revealed that the serum-free culture mediumsupplemented with ITS and bFGF had a very strong cell protecting actionand exhibited a unique property of inducing the proliferation ofcardiomyocytes. Hence, Example 8 was performed in order to show whatactions bFGF and insulin, a component of the ITS solution, would have onthe increase in the diameter of cell masses.

Basically, experiments were conducted as in Example 7, except that thefollowing culture media were used: α-MEM alone; α-MEM+50 ng/ml bFGF;α-MEM+10 μg/ml insulin+5 ng/ml bFGF; and α-MEM+1 μg/ml insulin+1 ng/mlbFGF.

Six days later, the cell masses of cardiomyocytes derived from mouseembryonic stem cells were observed; as the result, in each of α-MEM+50ng/ml bFGF, α-MEM+30 10 μg/ml insulin+5 ng/ml bFGF, and α-MEM+1 μg/mlinsulin+1 ng/ml bFGF, a significant increase in the diameter of cellmasses was seen as compared to the cell mass in the culture mediumconsisting of α-MEM alone (FIG. 7B). In addition, the effect oncontractile activity of the cardiomyocytes was strong and the same aswhat was achieved when ITS was added in Example 7.

From the foregoing, it is believed that the serum-free culture mediumsupplemented with bFGF or insulin+bFGF has a very strong cell protectingaction and exhibits a unique property of inducing the proliferation ofcardiomyocytes.

Example 9 Actions of “Serum-Free” and bFGF in Plane Adhesive CultureSystem of Purified Cardiomyocytes Derived from Mouse Embryonic StemCells

Cardiomyocytes derived from mouse embryonic stem cells were purified inaccordance with Example 1. The purified cardiomyocytes were seeded inthe same numbers on fibronectin-coated cell culture dishes and subjectedto plane adhesive culture in a variety of culture media. The variousculture media all comprised α-MEM as a basal culture medium but theyrespectively had the following components added thereto: 10% FBS alone,10% FBS+50 ng/ml bFGF, 10% KSR (knockout serum replacement: Invitrogen),ITS, and ITS+50 ng/ml bFGF. The cells seeded under those conditions werecultured for a total of 5 days and then photographed (FIG. 8A). As itturned out, the serum-free ITS and ITS+bFGF groups had significantlylower cell viability than the groups added with 10% FBS or 10% KSR.

Example 10 Transplantation of Purified Cardiomyocytes Derived from MouseEmbryonic Stem Cell into Myocardial Tissue of Immunodeficient Rat andMeasurement of Their Engraftment Rate

To begin with, the following experiment was conducted in order tomeasure the survival rate of purified, cardiomyocytes derived from mouseembryonic stem cell for the case where the reaggregation method was notapplied.

A total of 2×10⁵ cells were transplanted into the left ventricular freewall of an immunodeficient mouse (NOD-SCID). Anesthesia was induced onthe mouse with ether and maintained using air containing 2% isofluranesupplied through an artificial respirator. The mouse was subjected tothoracotomy (in the third intercostal space) under deep anesthesia andthe cardiac sac was ruptured with tweezers to expose the heart.Physiological saline (30 μl) containing cell masses of cardiomyocyteswas injected through a syringe with a 30 G needle. For injection, theneedle was inserted into the cardiac apex, from which it was advancedthrough the cardiac free wall by approximately 3 mm toward the cardiacbase. After the transplantation, the chest was closed quickly and, afterthe recovery of spontaneous beating, the mouse was returned into thecage.

Three weeks after the transplantation, the heart was fixed underperfusion and frozen sections were prepared. The sections wereimmunostained with an anti-sarcomeric actinin antibody and fluorescentmicroscopic images were taken (FIG. 9A). As it turned out, viable celltransplants were barely seen and only the reaction with the tracer reddye could be found (FIGS. 9A and 9D).

In the next place, cell masses each consisting of 2000 cardiomyocytes asconstructed under the serum-free conditions described in Example 7 (atotal number of 2×10⁵ cells) were transplanted into the left ventricularfree wall of an immunodeficient mouse (NOD-SCID). The transplantationwas carried out as in the experiment described above and 3 weeks later,the heart was fixed under perfusion and frozen sections were prepared.The sections were immunostained with an anti-sarcomeric actinin antibodyand fluorescent microscopic images were taken (FIG. 10). Based on thefluorescent microscopic images, the number of cells engrafted on thehost cardiac tissue contained in one cell mass was counted.

As it turned out, assuming that each of the transplanted cell massesaccurately consisted of 2000 cardiomyocytes, 92.05±11.1% cardiomyocyteswere found engrafted (n=4) (FIGS. 11A and 11B). In contrast, when thepurified disaggregated cells were injected as such (as dispersed), noengrafted cells could be found. This result means that thepost-transplantation engraftment rate of cardiomyocytes dramaticallyimproved from 0% to 92%.

Further, with a view to verifying the change in cardiomyocytes duringlong-term transplantation, investigation based on immunostaining of theheart was performed 3 and 8 weeks after the transplantation. As itturned out, the cytoplasm volume of cardiomyocytes increased markedly 3and 8 weeks after the transplantation, as compared with thecardiomyocytes before the transplantation (“Pre” in FIG. 11C), and whatis more, the transplanted cardiomyocytes aligned in the same directionas the cardiomyocytes in the host (FIGS. 11C and 11D). This shows thatthe transplanted cell masses of cardiomyocytes matured in the hostcardiac tissue.

Example 11 Preparation of Cell Masses Using Purified CardiomyocytesDerived from Marmoset Embryonic Stem Cells

The purpose of this Example was to prepare cell masses of purifiedcardiomyocytes derived from marmoset embryonic stem cell underserum-free conditions either with the addition of ITS or KSR.

Briefly, cell masses of purified cardiomyocytes derived from marmosetembryonic stem cells were prepared in accordance with Example 3,provided that cell masses were cultured in a serum-free culture mediumalone (FIG. 12A) or a serum-free culture medium supplemented with 10%KSR (FIG. 12B) or ITS (FIG. 12C). The cell masses constructed either 12hours or 3 days after the start of transplantation are shown in FIG. 12.

Example 12 Construction of “Thick” Cell Sheets Using Cell Masses ofPurified Cardiomyocytes Derived from Marmoset Embryonic Stem Cells

The cell masses of purified cardiomyocytes derived from marmosetembryonic stem cell that were prepared in Example 11 were suspensioncultured in the same plane. With the lapse of time over the period offrom 0 to 12 hours, adjacent cell masses are conjugated together to forma “thick” cell sheet of cardiomyocytes. Example 12 describes a modelexperiment intended to demonstrate the applicability of the method ofthe present invention. In actual application embodiments, cell masses ina desired size of purified cardiomyocytes derived from embryonic stemcells can be used in a desired number to construct a cell sheet of adesired size (FIG. 13). In addition, a cell sheet of a desired thicknesscan be formed depending on the size of the cell masses to be used.

Example 13 Transplantation of Cardiomyocytes Derived from HumanEmbryonic Stem Cell to the Immunodeficient Mouse Heart

In this Example, experiments were to determine whether the cell massesof cardiomyocytes which were prepared by differentiating human embryonicstem cells into cardiomyocytes would have the ability to be engrafted inthe cardiac tissue.

The human embryonic stem cells were obtained from the Stem Cell ResearchCenter, adjunct facilities to the Institute for Frontier MedicalSciences, Kyoto University (the Embryonic Stem Cell Center sponsored bythe National Bio-resource Project).

Using mouse embryonic fibroblasts (MEF) that had been growth-inactivatedby mitomycin C treatment, these human embryonic stem cells were culturedsuch that they would remain undifferentiated. The culture medium wascomposed of F12/DMEM (1:1) (SIGMA, Lot No. D6421), 20% KO-SERUM (GIBCO),1.6 mM L-glutamine, 0.1 mM non-essential amino acids (MEM), 0.1 mMβ-mercaptoethanol (2-ME; Sigma), 100 IU/ml penicillin, 100 μg/mlstreptomycin sulfate, and a recombinant human basic fibroblast growthfactor (bFGF; Peprotech). For serial passage, colonies of embryonic stemcells were separated by treatment with 0.1% type III collagenase(Wortington) at 37° C. for 10 minutes.

Subsequently, in order to separate the embryonic stem cells from MEF,the culture medium containing cell masses was passed through a mesh witha pore size of 40 μm and the cell masses in the oversize fraction wererecovered. The recovered cell masses were those of pure embryonic stemcells. For differentiation, 50-1,000 embryonic stem cells per EB werecultured as embryoid bodies on a non-cell-adhesive bacterium dish (AsahiTechno Glass; sterile Petri dish) for a total of 15-30 days so that theydifferentiated into embryoid bodies including cardiomyocytes. Theculture medium used for this differentiation was the same as identifiedabove, except that it did not contain bFGF, i.e., it was composed ofF12/DMEM (1:1) (SIGMA, Lot No. D6421), 20% KO-SERUM (GIBCO), 1.6 mML-glutamine, 0.1 mM non-essential amino acids (MEM), 0.1 mMβ-mercaptoethanol (2-ME; Sigma), 100 IU/ml penicillin, and 100 μg/mlstreptomycin sulfate.

Cardiomyocytes derived from human embryonic stem cells were purified inaccordance with Example 1. Then, in accordance with the results ofExample 8, cell masses each containing 1000 purified cardiomyocytes wereprepared using a serum-free α-MEM solution that contained 1 μg/mlinsulin and 1 ng/ml bFGF (see FIG. 14).

Further, the cell masses were transplanted into the cardiac tissue of animmunodeficient mouse in accordance with Example 10. Two weeks after thetransplantation, frozen sections were prepared in accordance withExample 10. The thus prepared sections were immunostained with Nkx2.5and an anti-sarcomeric actinin antibody and fluorescent microscopicimages were obtained.

Some cell masses were found to be stained with the red dye used as atracer of the transplanted cells. The sections were detected for theNkx2.5 and anti-sarcomeric actinin antibody by an immunological method.The result is shown in FIG. 15. The transplanted cells, being stainedwith the red tracer dye, were shown to be predominantly red-colored.Immunostaining with actinin also showed the staining of the striation.It was also shown that Nkx2.5, a marker of cardiomyocytes, waspredominantly found in the nuclei of the transplanted cardiomyocytes.This is a phenomenon peculiar to immature cardiomyocytes. In addition,the nuclei of cardiomyocytes derived from human embryonic stem cells arelarger than surrounding mouse cardiomyocytes and, hence, can bedistinguished from the latter (FIG. 15).

Further, 5 weeks after the transplantation, frozen sections wereprepared in accordance with Example 10. Some cell masses were found tobe stained with the red dye used as a tracer of the transplanted cells(FIG. 16). The thus prepared sections were immunostained with Nkx2.5(watery blue) and an anti-sarcomeric actinin antibody (green) or withNkx2.5 (watery blue) and an anti-human nuclear antigen (green: bindingto an antigen that was not present in mouse nuclei but present only inprimates) and fluorescent microscopic images were obtained. The resultsare shown in FIGS. 17 and 18. The mitochondria in the transplanted cellswere stained with the tracer dye, allowing the red dye to be detected asspots. Immunostaining with actinin also showed the staining of thestriation (FIG. 17). To show that these cell populations composing thesame region were derived from human cells, detection was performed usingan anti-human antibody and again it was demonstrated that thetransplanted cells were human cells, in particular, cardiomyocytes thatwere co-stained with Nkx2.5 (FIG. 18).

Example 14 Preparation of Cell Masses Using Purified CardiomyocytesDerived from Mouse Induced Pluripotent Stem (iPS) Cells

The purpose of this Example was to prepare cell masses of purifiedcardiomyocytes derived from mouse iPS cells under serum-free conditionswith or without addition of ITS or KSR.

The mouse iPS cells were assigned from the Institute for FrontierMedical Sciences, Kyoto University. Differentiation of the mouse iPScells into cardiomyocytes was carried out as in Example 1. In thatinstance, it was found to be optimum that 1000 cells were used as theinitial cells for composing one embryoid body.

FIG. 19A shows the result of an FACS analysis conducted with themitochondrial indicator TMRM in order to purify cardiomyocytes. Therectangle in the graph represents the region of cardiomyocytes. The thuspurified cardiomyocytes were subjected to adhesive culture andimmunostained (with actinin and Nkx2.5); the result is shown in FIG.19B. Since the mouse iPS-induced cardiomyocytes were shown to formaggregated cell masses, it was revealed that the cell fractionsrecovered by FACS consisted of nearly 100% of cardiomyocytes. Further,FIG. 19C shows the appearance of the purified cardiomyocytes 24 hoursafter they were seeded in a non-cell-adhesive 96-well culture dish. Inthis case, a serum-free culture medium was used. As it turned out, thepurified cardiomyocytes derived from mouse iPS cells could also be usedto construct cell masses by the method of the present invention.

Example 15 Culture Medium Composition Optimum for Forming Cell MassesUsing Cardiomyocytes Derived from Human Embryonic Stem Cells

In Example 7, it was revealed that the serum-free culture mediumsupplemented with ITS and bFGF had a very strong protecting action onmouse-derived cells and exhibited a unique property of inducing theproliferation of cardiomyocytes. Hence, Example 15 was carried out inorder to show the effectiveness of bFGF in cardiomyocytes derived fromhuman ES cells and to review its effectiveness more closely by comparingit with other growth factors.

Basically, an α-MEM+ITS was used as a culture medium. This basal culturemedium was supplemented with 25 ng/ml bFGF (Peprotech, Inc., Rocky Hill,N.J., USA), 25 ng/ml acidic FGF (aFGF), 25 ng/ml FGF-4, 20 ng/mlkeratinocyte growth factor (KGF), 100 ng/ml stem cell factor (SCF), 100ng/ml vascular endothelial growth factor (VEGF), 10 ng/ml leukemiainhibiting factor (LIF) (Millipore Corporation, Billerica, Mass., USA),100 ng/ml glial cell line-derived neurotrophic factor (GDNF), 20 ng/mlhepatocyte growth factor (HGF), 10 ng/ml insulin-like growth factor(IGF)-1, 100 ng/ml epidermal growth factor (EGF), 1×10⁻⁷ M endothelin-1(ET-1), 10 ng/ml platelet derived growth factor (PDGF)-AA, or 100 ng/mlPDGF-BB (those reagents without the indication of where to obtain wereall purchased from R&D systems). Human ES cells were cultured using eachof the culture medium to prepare cell aggregates.

The diameter of cell masses was measured 3, 8, 25 and 40 days after thepreparation of cell masses. As it turned out, the cell mass prepared inthe presence of bFGF had the largest diameter on each of the days (FIG.20A). It was also found that this protecting action was continued aslong as 40 days. Instead of bFGF, each of the various substancesmentioned above was added to the culture medium and checked for theireffect on the formation of cell masses; EGF, PDFG-BB and ET-1 were foundto be effective, though not as effective as bFGF (FIG. 20B).

As a result, it turned out that, even in the case of differentiation ofcardiomyocytes derived from human ES cell, bFGF has cell protecting andgrowth promoting activities under serum-free conditions and that theseactions are stronger than those of other growth factors.

Further, in order to elucidate the mechanism by which cardiomyocytestransplanted into the host heart can mature in the cardiac tissue aftertransplantation, bFGF, EGF, PDGF-BB and ET-1 were tested by real-timePCR (Applied Biosystems) for the possibility of gene expression in thehost heart. The primers and probes for the respective genes werepurchased from Applied Biosystems (TaqMan gene expression assays); to bemore specific, bFGF (Mm0128715_m1), EGF (Mm01316967_m1), PDGF-BB(Mm01298577_m1), and ET-1 (Mm01351840_g1) were used. The reagents usedfor analysis and the operating procedure were in accordance with theinstruction manual provided by Applied Biosystems. As a result, itturned out that the genes mentioned above were expressed in the hostheart (FIG. 21).

The results of Example 15 suggested that the group of growth factorsrequired for the survival and maturation of the cell masses ofcardiomyocytes transplanted into the heart are supplied from the hostheart.

Industrial Applicability

According to the present invention, it has been found thatcardiomyocytes derived from embryonic stem cell that have been purifiedby dispersing to single cells have such a new characteristic that theyare capable of aggregating when they are cultured under serum-freeconditions. By constructing cell masses using the method of the presentinvention, long-term culture can be performed with the survival rate orproliferative capacity of those cardiomyocytes being maintained at highlevels. It has further been found that, when those cells aretransplanted to the cardiac tissue of an individual (the living body),their engraftment rate in the cardiac tissue is significantly enhanced,with the result that the cardiomyocytes will not mix withnon-cardiomyocytes but can be made engrafted for an extended period oftime within the cardiac tissue. Thus, the present invention has enhancedthe feasibility of providing cardiomyocytes for transplantation, as wellas a method of cell therapy on the heart which is alternative to cardiactransplantation as a treatment of cardiac disease by transplantingcardiomyocytes that have been prepared outside the living body, and amedical device comprising cell masses of cardiomyocytes.

The invention claimed is:
 1. A method of preparing cell masses ofcardiomyocytes derived from pluripotent stem cells, wherein the cellmasses of cardiomyocytes derived from pluripotent stem cells areobtained by culturing a purified fraction of single cells ofcardiomyocytes derived from pluripotent stem cells in a culture mediumunder serum-free conditions in suspension culture conditions in around-bottomed well so that they are aggregated to form cell masses ofcardiomyocytes derived from pluripotent stern cells, wherein thepurified fraction of single cells of cardiomyocytes are obtained byinducing differentiation of pluripotent stem cells into cardiomyocytesresulting to form embrovid bodies, dispersing the embryoid bodies andpurifying a fraction of single cells of cardiomyocytes derived frompluripotent stem cells.
 2. The method according to claim 1, wherein thepluripotent stem cells are selected from the group consisting ofembryonic stem cells, embryonic germ cells, germnline stem cells, andinduced pluripotent stem cells.
 3. The method according to claim 1,wherein the culture medium contains insulin.
 4. The method according toclaim 1, wherein the culture medium contains at least one substanceselected from the group consisting of transferrin, selenium, a basicfibroblast growth factor (bFGF), an epidermal growth factor (EGF), aplatelet-derived growth factor-BB (PDGF-BB), and endothelin-1 (ET-1). 5.The method according to claim 1, wherein the content in the culturemedium is 0.1 to 10 mg/L of insulin, 0.1 to 10 mg/L of transferrin, 0.1to 10 μg/L of selenium, 1 ng/ml to 100 ng/ml of the basic fibroblastgrowth factor, 1 ng/ml to 1000 ng/ml of the epidermal growth factor, 1ng/ml to 1000 ng/ml of the platelet-derived growth factor, and 1×10⁻⁸ to1×10⁻⁶ M of endothelin-1 (ET-1).
 6. A method of treating cardiacdisease, characterized in that cell masses of cardiomyocytes derivedfrom pluripotent stem cells are transplanted to the cardiac tissue of anindividual such that they are engrafted, wherein the cell masses ofcardiomyocytes derived from pluripotent stem cells are obtained byculturing a purified fraction of single cells of cardiomyocytes derivedfrom pluripotent stem cells in a culture medium under serum-freeconditions in suspension culture conditions in a round-bottomed well sothat they arc aggregated to form cell masses of cardiomyocytes derivedfrom pluripotent stem cells, and wherein the purified fraction of singlecells of cardiomyocytes are obtained by inducing differentiation ofpluripotent stem cells into cardiomyocytes resulting to form embrovidbodies, dispersing the embryoid bodies and purifying a fraction ofsingle cells of cardiomyocytes derived from pluripotent stem cells. 7.The method according to claim 6, wherein the pluripotent stem cells areselected from the group consisting of embryonic stem cells, embryonicgerm cells, germline stem cells, and induced pluripotent stem cells. 8.The method according to claim 6, wherein the transplantation comprisesinjecting the cell masses of cardiornyocytes into the cardiac tissue. 9.The method according to claim 6, wherein the transplantation comprisestransplanting a sheet of cell masses of cardiomyocytes onto the cardiactissue.
 10. A method of preparing a sheet of cell masses ofcardiornyocytes, characterized in that cell masses of eardiomyocytesderived from pluripotent stem cells are seeded on the surface of awall-partitioned, non-cell-adhering vessel, with no space between cellmasses such that adjacent cell masses will be continuously in contactwith each other, followed by suspension culture which is maintaineduntil the cell masses are conjugated together to have a desiredthickness of 50-300 μm, wherein the cell masses of cardiomyocytes areobtained by culturing a purified fraction of single cells ofcardiomyocytes derived from pluripotent stem cells in a culture mediumunder serum-free conditions in suspension culture conditions in around-bottomed well so that they are aggregated to form cell masses ofcardiomyoutes derived from pluripotent stem cells, and wherein thepurified fraction of single cells of cardiomyocytes are obtained byinducing differentiation of pluripotent stem cells into cardiomyocytesresulting to form embrovid bodies, dispersing the embryoid bodies andpurifying a fraction of single cells of cardiomyocytes derived frompluripotent stem cells.
 11. The method according to claim 10, whereinthe pluripotent stem cells are selected from the group consisting ofembryonic stern cells, embryonic germ cells, germline stern cells, andinduced pluripotent stem cells.
 12. A medical device for transplantationto the cardiac tissue of an individual having a cardiac disease, themedical devise comprising cell masses of cardiomyocytes derived frompluripotent stem cell, wherein the cell masses of cardiomyocytes areobtained by culturing a purified fraction of single cells ofcardiomyocytes derived from pluripotent stem cells in a culture mediumunder serum-free conditions in suspension culture conditions in around-bottomed well so that they are aggregated to form cell masses ofcardiomyocytes derived from pluripotent stem cells, and wherein thepurified fraction of single cells of cardiomyocytes are obtained byinducing differentiation of pluripotent stem cells into cardiomyocytesresulting to form embrovid bodies, dispersing the embryoid bodies andpurifying a fraction of single cells of cardiomyocytes derived frompluripotent stem cells.
 13. The medical device according to claim 12,wherein the pluripotent stem cells are selected from the groupconsisting of embryonic stem cells, embryonic germ cells, germline stemcells, and induced pluripotent stem cells.
 14. The medical deviceaccording to claim 12, wherein the transplantation comprises injectingthe cell masses of cardiomyocytes into the cardiac tissue.
 15. Themedical device according to claim 12, wherein the transplantationcomprises transplanting a sheet of the cell masses of cardiomyocytesonto the cardiac tissue.